DNA fragmentation in epididymal freeze-dried ram spermatozoa impairs embryo development

Abstract

Sperm freeze-drying is a revolutionary technique, which has been gaining prominence in recent years. The first related significant result was Wakayama and Yanagimachi’s demonstration in 1998 of the birth of healthy mouse offspring by Intracytoplasmic Sperm Injection (ICSI), using epididymal freeze-dried spermatozoa. Mouse, rat, and hamster models were the first small mammals born from lyophilized epididymal spermatozoa, whereas most other studies in this field used ejaculated spermatozoa. In this work, we applied this technique to ram epididymal spermatozoa, checking the correlation between DNA integrity and embryo development following ICSI. To do this, epididymal sperm from four rams was lyophilized in a trehalose, glucose, KCl, HEPES, and Trolox media. To evaluate DNA damage and fragmentation after rehydration, samples were processed for Sperm Chromatin Dispersion test (SCD), Two-Tailed Comet Assay, and were used for ICSI. Ram #2 had a higher rate of spermatozoa with intact DNA compared with rams #1, #3, and #4 (28% vs. 3.8%, 2.8%, and 5%, respectively) and the lowest rate of Single-Strand Breaks (SSBs) (70% vs. 95.9%, 92.6%, and 93% respectively). Ram #3 had a higher level of Double-Strand Breaks (DSBs) compared to Ram #1 (4.6% vs. 0.33%, respectively). Embryo development to the blastocyst stage following ICSI was only reached from rams whose sperm had higher level of intact DNA – Rams #2 and #4 (6%, 5/147 and 6.3%, 4/64, respectively). Definitively, the impact of sperm DNA damage on embryonic development depends on the balance between sperm DNA fragmentation extent, fragmentation type (SSBs or DSBs), and the oocyte’s repair capacity.

Desiccation and supra‐zero temperature storage of cat germinal vesicles lead to less structural damage and similar epigenetic alterations compared to cryopreservation

“…Some cryoprotectants, such as dimethyl sulfoxide (DMSO) used in this study, have been linked to increased abnormal chromatin despite their function in mitigating freezing damages Hu, Marchesi, Qiao, & Feng, 2012). During desiccation, DNA is also susceptible to hyperosmotic and dehydration stress (Palazzese, Gosálvez, Anzalone, Loi, & Saragusty, 2018;Shahba et al, 2016).…”

“…Some cryoprotectants, such as dimethyl sulfoxide (DMSO) used in this study, have been linked to increased abnormal chromatin despite their function in mitigating freezing damages (Falk et al, ; Hu, Marchesi, Qiao, & Feng, ). During desiccation, DNA is also susceptible to hyperosmotic and dehydration stress (Palazzese, Gosálvez, Anzalone, Loi, & Saragusty, ; Shahba et al, ). Results in the present and previous studies indicate that current microwave‐assisted dehydration protocol with the use of trehalose can effectively protect DNA integrity and chromatin conformation in the majority of GVs (Elliott et al, ).…”

Exploring dry storage as an alternative biobanking strategy inspired by Nature

“…So, it is clear that there is some level of deterioration along the way from the testis out. The only studies to date performed on drying epididymal spermatozoa from large mammals include two reports on cat spermatozoa [88,89], and a study on ram spermatozoa [90] in which the importance of single-strand DNA breaks (in adition to doublestrand breaks) was demonstrated. While a number of studies have evaluated DNA integrity, this seem to be the only one in which single-strand breaks were evaluated.…”

“…80,98,99] while in other studies an attempt to obtain viable cells was made by using proper cryopreservation techniques [e.g. 90,100]. Does this suggest that most researchers approach the field with the assumption that spermatozoa would not survive the process and that as long as the DNA remains relatively intact, these cells can be used in ICSI?…”

Effect of chelating and antioxidant agents on morphology and DNA methylation in freeze‐drying rabbit (Oryctolagus cuniculus) spermatozoa

“…However, FD method may affect negatively spermatozoa not only by altering their morphology membrane and acrosome, but also by causing molecular damages on DNA and centrosome, due to snap‐freezing and drying stresses (Liu et al, ). Among all these sperm aberrations, DNA fragmentation of spermatozoa following FD is one of the main causes of embryonic development failure (Palazzese, Gosálvez, Anzalone, Loi, & Saragusty, ). In particular, endonucleases may induce oxidative stress due to the release of reactive oxygen species (ROS) (Kusakabe, Szczygiel, Whittingham, & Yanagimachi, ).…”